Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.
Rapid Commun Mass Spectrom. 2013 Nov 15;27(21):2453-60. doi: 10.1002/rcm.6708.
25-Hydroxyvitamin D3 [25(OH)D3] is the best-established indicator of vitamin D status. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD), a representative Cookson-type reagent, has often been employed for enhancing the sensitivity in the trace determination of 25(OH)D3 in a neonatal dried blood spot (DBS), which contains only 2.65 μL of whole blood, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The objective of this study was the development of a novel Cookson-type reagent surpassing PTAD in terms of sensitivity and specificity in the LC/ESI-MS/MS assay of 25(OH)D3.
A novel Cookson-type reagent, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), was synthesized from 4-dimethylaminobenzoyl chloride. The DAPTAD-derivative of 25(OH)D3 was prepared and its LC/ESI-MS/MS behavior was examined. The applicability of the DAPTAD-derivatization in the determination of 25(OH)D3 in neonatal DBSs was also examined.
The derivatization was completed at room temperature within 1 h. The DAPTAD-derivative of 25(OH)D3 provided a characteristic product ion derived from the cleavage of the vitamin D skeleton during MS/MS. The limit of detection of the DAPTAD-derivative during selected reaction monitoring was 0.25 fmol on the column, which was 30 and 2 times lower than those of the intact 25(OH)D3 and the PTAD-derivative, respectively. The DAPTAD-derivatization followed by LC/ESI-MS/MS enabled the detection of a trace amount (in the low-ng/mL range) of 25(OH)D3 in DBSs with a simple pretreatment (only methanol extraction) and short chromatographic run time (10 min). The DAPTAD-derivatization was also useful for the separation of 25(OH)D3 from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3]. On the contrary, the assay using the PTAD-derivatization might lead to overestimation of the true 25(OH)D3 levels due to the co-elution of 25(OH)D3 and 3-epi-25(OH)D3.
We developed DAPTAD for enhancing the sensitivity and specificity of the LC/ESI-MS/MS assay of 25(OH)D3. Our new method using DAPTAD can reduce the overestimation of the 25(OH)D3 levels, and will prove helpful in the diagnosis of vitamin D deficiency in infants.
25-羟维生素 D3（25（OH）D3）是维生素 D 状态的最佳指标。4-苯-1,2,4-三唑啉-3,5-二酮（PTAD）是一种代表性的 Cookson 型试剂，常用于增强痕量 25（OH）D3 在新生儿干血斑（DBS）中的检测灵敏度，DBS 中仅含有 2.65μL 的全血，使用液相色谱/电喷雾串联质谱法（LC/ESI-MS/MS）。本研究的目的是开发一种新型的 Cookson 型试剂，在 25（OH）D3 的 LC/ESI-MS/MS 分析中，其灵敏度和特异性均优于 PTAD。
通过 4-二甲基氨基苯甲酰氯合成了一种新型的 Cookson 型试剂 4-（4'-二甲氨基苯基）-1,2,4-三唑啉-3,5-二酮（DAPTAD）。制备了 25（OH）D3 的 DAPTAD 衍生物，并对其 LC/ESI-MS/MS 行为进行了考察。还考察了 DAPTAD 衍生化在新生儿 DBS 中 25（OH）D3 测定中的适用性。
室温下 1 小时内完成衍生化。25（OH）D3 的 DAPTAD 衍生物在 MS/MS 中提供了源自维生素 D 骨架断裂的特征产物离子。在选择反应监测中，DAPTAD 衍生物的检测限为 0.25 fmol 柱上，分别比完整的 25（OH）D3 和 PTAD 衍生物低 30 倍和 2 倍。通过 LC/ESI-MS/MS 进行 DAPTAD 衍生化，可在简单预处理（仅甲醇提取）和较短的色谱运行时间（10 分钟）下检测到 DBS 中痕量（低 ng/mL 范围）的 25（OH）D3。DAPTAD 衍生化对于分离 25（OH）D3 和强代谢物 3-表-25-羟基维生素 D3（3-epi-25（OH）D3）也很有用。相反，由于 25（OH）D3 和 3-epi-25（OH）D3 的共洗脱，使用 PTAD 衍生化的测定可能导致对 25（OH）D3 真实水平的高估。
我们开发了 DAPTAD 以增强 25（OH）D3 的 LC/ESI-MS/MS 分析的灵敏度和特异性。我们使用 DAPTAD 的新方法可以减少对 25（OH）D3 水平的高估，这将有助于婴儿维生素 D 缺乏症的诊断。
